The Dynamic Emergence of GATA1 Complexes Identified in in Vitro Embryonic Stem Cell Differentiation and in Vivo Mouse Fetal Live

ARTICLE Hematopoiesis The dynamic emergence of GATA1 complexes Ferrata Storti Foundation identified in in vitro embryonic stem cell differentiation and in vivo mouse fetal liver

Xiao Yu,1,2 Andrea Martella,1,3 Petros Kolovos,1,4 Mary Stevens,1 Ralph Stadhouders,1,5 Frank G. Grosveld1 and Charlotte Andrieu-Soler1,6,7 1Department of Cell Biology, ErasmusMC, Rotterdam, the Netherlands; 2Current address: Department of Medical Microbiology, Amsterdam University Medical Center, Amsterdam, Haematologica 2020 the Netherlands; 3AstraZeneca, R&D Innovative Medicines, Cambridge Science Park, Volume 105(7):1802-1812 Milton Road, Cambridge, UK; 4Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, Denmark; 5Department of Pulmonary Medicine, Erasmus MC, Rotterdam, the Netherlands; 6Institut de Génétique Moléculaire Montpellier, Université de Montpellier, CNRS, Montpellier, France and 7Université de Paris, Laboratoire d’excellence (LabEx) du globule rouge GR-Ex, Paris, France

ABSTRACT

ATA1 is an essential transcriptional regulator of myeloid hematopoietic differentiation towards red blood cells. During ery- Gthroid differentiation, GATA1 forms different complexes with other transcription factors such as LDB1, TAL1, E2A and LMO2 (“the LDB1 complex”) or with FOG1. The functions of GATA1 complexes have been studied extensively in definitive erythroid differentiation; however, the temporal and spatial formation of these complexes during erythroid development is unknown. We applied proximity ligation assay (PLA) to detect, localize and quantify individual interactions during embryonic stem cell differentiation and in mouse fetal liver (FL) tissue. We show that GATA1/LDB1 interactions appear before the proerythroblast stage and increase in a subset of Correspondence: the CD71+/TER119– cells to activate the terminal erythroid differentiation CHARLOTTE ANDRIEU-SOLER program in 12.5 day FL. Using Ldb1 and Gata1 knockdown FL cells, we [email protected] studied the functional contribution of the GATA1/LDB1 complex during differentiation. This shows that the active LDB1 complex appears quite late Received: January 7, 2019. at the proerythroblast stage of differentiation and confirms the power of PLA in studying the dynamic interaction of proteins in cell differentiation at Accepted: October 3, 2019. the single cell level. We provide dynamic insight into the temporal and spa- Pre-published: October 3, 2019. tial formation of the GATA1 and LDB1 transcription factor complexes during hematopoietic development and differentiation. doi:10.3324/haematol.2019.216010

Check the online version for the most updated information on this article, online supplements, Introduction and information on authorship & disclosures: www.haematologica.org/content/105/7/1802 The first hematopoietic cells appear in yolk sac blood islands on embryonic day 6.5 (E6.5) during mouse development. On E10.5 to E11, definitive hematopoietic stem cells (HSC) appear in the aorta-gonad-mesonephros (AGM) region within the ©2020 Ferrata Storti Foundation embryo (and the vitelline and umbilical arteries). They migrate to the fetal liver Material published in Haematologica is covered by copyright. (FL), mature from pre-HSC to HSC, and after moving, reside in the adult bone mar- All rights are reserved to the Ferrata Storti Foundation. Use of row.1,2 One of the lineages originating from HSC generates erythroid cells. published material is allowed under the following terms and conditions: GATA1 is one of the essential transcription factors for the erythroid (and -/- https://creativecommons.org/licenses/by-nc/4.0/legalcode. megakaryocytic) program. Gata1 knockouts (KO) (Gata1 ) die between E9.5 to E10 Copies of published material are allowed for personal or inter- due to a block of differentiation at the proerythroblast stage, leading to the absence nal use. Sharing published material for non-commercial pur- of mature red blood cells.3,4 GATA1 can form several complexes to regulate ery- poses is subject to the following conditions: 5 https://creativecommons.org/licenses/by-nc/4.0/legalcode, throid gene expression. Two proteins of particular interest bind directly to GATA1. sect. 3. Reproducing and sharing published material for com- The first, FOG1 (Friend of GATA1), binds to the N-terminal zinc finger (ZnF) of mercial purposes is not allowed without permission in writing GATA1 and recruits the chromatin remodeling complex NuRD/MeCP1 and/or the from the publisher. C-terminal binding protein (CTBP) corepressor-containing complex to regulate GATA1 target genes.6 The second is LMO2, which is part of a larger complex7-9 containing the LIM-domain-binding protein 1 (LDB1). LDB1 functions as a scaffold

1802 haematologica | 2020; 105(7) Temporal and spatial emergence of GATA1 complex protein to form multiprotein transcription complexes that RNA interference regulate the differentiation of various cell types. Ldb1 KO Lentiviral particles for LDB1 were produced as described by (Ldb1-/-) mice die between E9.5 and E10 due to severe Stadhouders R15 using Ldb1 shRNA (shRNA#1: 5’-GGACCAAA- defects in a number of developing tissues, including GAGATATACCA-3’, shRNA#2: 5’-GACTCTGTGTGATACTA- abnormal hematopoietic development.10 This abnormal GA-3’) and Gata1 shRNA (5’-GTTTGGATGCAGCATCTTCTT- hematopoiesis is also observed in knockout mouse 3’) with non-targeting shRNA as controls. Lentiviral infected cells embryos lacking the LDB1 binding-partners TAL111 or were harvested 72 hours after transduction and processed for LMO2.12 nuclear extraction. Despite the knowledge on GATA1 binding partners, it is not known when and where GATA1 complexes form. In Protein analysis order to identify the temporal and spatial appearance of Murine erythroleukemia (MEL) cells or EB nuclear extract and GATA1/FOG1 and GATA1/LDB1 complexes during differ- immunoprecipitation (IP) were prepared as described16 and size- entiation, we applied proximity ligation assays (PLA)13 in exclusion chromatography was performed on an AKTA-FPLC differentiated mouse embryonic stem (ES) cells and FL apparatus with a Superose-6 10/30 column (Amersham cells. We detect the first significant GATA1/LDB1 interac- Biosciences). Fractions were precipitated with trichloroacetic acid tion in CD71+ FL cells. Knockdown (KD) of LDB1 in vitro and analyzed by Western blotting using Odyssey system (LI- led to fetal cell death and decreased the CD71+ cell popu- COR). lations, providing functional evidence for its essential role at that stage of erythroid differentiation in normal FL. Immunofluorescent staining MEL or FL cells were stained as described8 and analyzed by confocal microscopy (Leica SP5). Methods PLA on EB and mouse embryo tissue Cell culture and mouse FL collection 10 μm sliced E4, 5, 9 EB or E12.5 mouse fetal tissues were fixed Wild-type (WT) and Ldb1-/- mouse ES cells were cultured in and processed for PLA following the manufacturer’s protocol DMEM-15% FCS-1% non-essential amino acids-100 units/mL (Duolink, OLINK) using antibodies indicated in the Online penicillin-100 mg/mL streptomycin-6.3e-4% 2-mercaptoethanol- Supplemntary Table S2. PLA signals were visualized by Leica SP5 100 units/mL Esgro. Day 12.5 (D12.5) or D13.5 FL were used for confocal microscopy and were analyzed using BlobFinder soft- cell sorting, nuclear extraction, or directly embedded in OCT ware (Uppsala University, Sweden). Signals contained in or adja- Tissue-Tek (Sakura) for tissue slicing. All animal experiments were cent to nuclei were compared between different groups (n=3). The performed according to guidelines and protocols that had been Kruskal–Wallis test for variance between groups was performed approved by an independent committee on the ethical use of and the Tukey method to counteract multiple comparison errors experimental animals (DEC). was applied. Deconvolution of PLA signals and volume analysis was performed using Huygens Suite as published.17 ES cell differentiation by the hanging drop method Mouse WT and Ldb1-/- ES cells were differentiated as described.14 On D4, D5 or D9 of ES cell differentiation, embryoid Results bodies (EB) were collected by flushing with PBS in 50 mL falcon tubes then embedded in the OCT Tissue-Tek. LDB1 complexes start to form at D4 of in vitro ES cell differentiation Flow cytometry analysis and cell sorting We applied PLA18 on sliced in vitro differentiated EB to Mouse E12.5 or E13.5 FL cells (infected or not by LDB1 or identify when GATA1 complexes form. This enables low GATA1 small hairpin RNA [shRNA]) were labeled with level detection of endogenous protein-protein interaction CD71-FITC and TER119-PE antibodies and sorted on a FACSAria in situ. III (BD Biosciences) into four populations: P1 (CD71–/TER119–), P2 First, we characterized gene expression dynamic for (CD71+/TER119–), P3 (CD71+/TER119+) and P4 (CD71–/TER119+). genes of interest during ES cell differentiation (Figure 1A). As expected, the stem cell marker Rex1 is expressed early Real-time quantitative PCR (RT-qPCR) (day 0 to 2 [D0-D2]) and decreases during differentiation, Total RNA was isolated from sorted FL cells or trypsin-dissoci- while β-globin increases at later stages at D5-D6. Thus ated EB up to D6 of differentiation with Trizol (Invitrogen). RT- Ldb1 is expressed both in early and late stages of ES differ- qPCR was performed using SybrGreen (Applied Biosystem) on entiation, and in the erythroid cell lineage at D5-D6. Bio-Rad CFX96. Rnh1 (ribonuclease inhibitor 1) gene was used as Following differentiation Gata1, Fog1, Gata2, Flk1, Tal1 internal control for normalization. Primers are indicated in the and Lmo2 expression gradually increases. Of note Gata2 Online Supplementary Table S1. gene induction starts at D4 whereas Gata1 expression is delayed for 24 hours (h) (Kolovos et al. submitted), i.e. the Gene expression profiling by RNA sequencing (RNA-seq) GATA-switch occurs in early embryogenesis.19 RNA samples from sorted mouse E12.5 FL cells, P1 to P4, were PLA representing combinations of transcription factor sequenced and analyzed as described10 using independent biolog- (TF) interactions (GATA1/LDB1, GATA1/FOG1 and ical replicates. Significant (at least ±0.6 log two-fold change and LDB1/E2A) was performed in undifferentiated cells D0, P-value ≤0.05) up- and down-regulated genes were selected. Data D4, D5 and D9 differentiated WT or Ldb1-KO EB (Figure are deposited in the Sequence Read Archive (SRA) (Accession 1B). Quantification of PLA signals showed that these inter- Number: SRP158286). actions are absent in ESC, while GATA1/LDB1 and LDB1/E2A interactions already occur at D4 of ES cell dif- Antibodies ferentiation. The GATA1/FOG1 interaction appeared 24 h Antibodies are indicated in the Online Supplementary Table S2. later at D5. No red blood cells emerged in Ldb1-KO EB at

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Figure 1. Gene expression and proximity ligation assay on embryonic stem cell differentiation. (AcGene expression during embryonic stem (ES) cell differentiation from day 0 until day 6 (D0-D6) of genes of interest. Standard error of the mean (SEM) from three biological replicates is shown as error bar. (B) Proximity ligation assay (PLA) detection of GATA1/FOG1, GATA1/LDB1 and LDB1/E2A complexes was performed on wild-type ES cell differentiation at D0 (ES cells), D4, D5 and D9, and Ldb1-KO ES cell differentiation at D9. On D9, EB from wild-type (WT) or Ldb1-KO are collected and pictured as shown. PLA signal representing protein-protein interaction was in white on a black background and in Texas-Red in merged pictures together with DAPI in blue. Each red dot represents a fluorescent signal of GATA1 complex formation. The images were analyzed using BlobFinder software38 which quantifies the number of PLA-positive dots in cells. The boxplot (Figure 1B, right panel) shows the density of the dots related to its nuclear area (from three biological replicates). Comparisons of the PLA signals obtained with two primary antibodies versus those obtained in the negative controls were tested in ANOVA and the significance is shown in asterisks. All scale bars represent 10 μm. PLA signals have been quantified on each day and compared with negative controls including GATA1, FOG1, or E2A single-antibody or secondary antibodies alone. Asterisk shows the significant interactions between the two primary antibodies PLA signal and single primary antibody controls. The significance was analyzed with Kruskal-Wallis test as follows, ****: P≤0.0001, ***: P≤0.001, **: P≤0.01, *: P≤0.05. The Tukey method to counteract multiple comparison errors was applied.

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D9 as shown by the lack of red coloration. This correlates signals were also detected for LDB1/LMO2, which is part with absence of interaction detection by PLA constituting of the same GATA1/LDB1 complex (Figure 2B) suggesting an important control for PLA specificity, in addition to the GATA1/LDB1 and LDB1/LMO2 and by inference the single probes and secondary antibodies alone used a neg- GATA1/LDB1/LMO2 complex are present at a high level ative control. in a subpopulation of cells. This is in accordance with our We next characterized the dynamic expression of previous co-immunoprecipitation and ChIP-sequencing hematopoietic TF proteins during the time course of ES co-localisation data of these factors in MEL and in Flk1+ cell differentiation at D4-5 using Ldb1-KO cells as the con- cells sorted four days after ES cell differentiation.5,8,10 In trol (Online Supplementary Figure S1A). This showed that contrast, GATA1/FOG1 signals appear in similar numbers the different factors already form a complex as deter- of cells but are less abundant/weaker and more evenly dis- mined by immunoprecipitation at D4 and D5 (Online tributed (Figure 2, upper panel A). In agreement with the Supplementary Figure S1C-D) using Ldb1-KO cells as the PLA results, immunofluorescent staining for individual control (Online Supplementary Figure S1E). Online LDB1, GATA1 and FOG1 proteins in FL sections showed Supplementary Figure S1C shows that LDB1 and FOG1 fail higher co-expression of GATA1 and LDB1 in a subpopula- to pull down GATA1 (and vice versa) in D4 differentiated tion of cells than GATA1 and FOG1 (Online Supplementary cells, which is likely due to very low amounts of the bridg- Figure S2), although it should be noted these are signals ing factor LMO2. Day 5 (Online Supplementary Figure S1D) from different antibodies (see Methods). shows more GATA1, but LMO2 is still undetectable and FL contains erythroid cells at different stages of differen- LDB1 and GATA1 appear to fail pulling down each other. tiation and the PLA results suggest that the LDB1 com- The same is seen for GATA1 and FOG1. Interestingly plex, is more important at particular stages of erythroid FOG1 appears to be regulated by (the) LDB1 (complex) as cells in agreement with data showing that GATA1 increas- it is present in D5 WT cells, but not in Ldb1-KO cells. This es before the end stage of erythroid differentiation8 prima- agrees with our observation that LDB1 binds to the Fog1 rily in the LDB1 complex. gene in Flk1 positive cells sorted four days after ES cell differentiation and the reduction of Fog1 expression in GATA1/LDB1 complex is highly localized in early Ldb1-KO cells analysed by RNA-Seq.10 The amounts of the erythroid differentiating cells in sorted FL proteins involved (directly or indirectly) were too low to In order to identify the cells containing high PLA signals, allow their detection by immunoprecipitation or mass E12.5 or E13.5 FL cells were sorted using glycophorin spectrometry (data not shown), because D4 and D5 differ- (TER119) and transferrin receptor (CD71) antibodies into entiated ES cells are a mixture of few hematopoietic cells four populations: P1 (CD71–/TER119–), P2 in the presence of many non-hematopoietic cells. For (CD71+/TER119–), P3 (CD71+/TER119+) and P4 example, many cells express Oct4 (Online Supplementary (CD71–/TER119+) in order to separate different stages Figure S1A) or cardiac genes in cardiac progenitors.20 The from proerythroblasts to orthochromatic erythroblasts21,22 complexes are barely detectable by size-exclusion chro- (Figure 3A). matography (Online Supplementary Figure S1B). By con- First, gene expression was measured in the four popu- trast, the clear PLA signals (Figure 1B) show the power of lations (Figure 3B). At early stages (P1 and P2), expression PLA to analyze complexes in individual cells. The few PLA of Ldb1, Gata1 and Fog1 starts increasing followed by a signals in undifferentiated ES cells are background since decrease at P3 for Ldb1 and at P4 for Gata1 and Fog1, such signals are also detected with single antibody con- which follow each other. Gata2, c-Kit and c-Myb genes are trols. expressed highly in P1 which contains precursor cells (and In summary, PLA monitors the dynamic changes of dif- other cell types), their expression decreases during differ- ferent protein complexes even of low amounts present in entiation, whereas the β-globin gene increased dramatical- a subset of cells. The PLA signals in D5 EB appear to dis- ly from P3 to P4. The CD71/TER119 sorted cells were tinguish a subpopulation of cells, suggesting that some used for RNA-seq analysis in two independent biological specification is already in progress towards hematopoietic replicates for each P1 to P4 population of E12.5 FL cells. cells in the mixed three-dimensional cell aggregates. Principal component analysis (Figure 3C) shows that biological replicates of each population cluster and can be The GATA1 complexes (GATA1/LDB1 and GATA1/FOG1) separated from each other. As expected, the RNA-seq are observed in mouse E12.5 FL cells result in those populations (Online Supplementary Figure S3) (Pre-)HSC move to the FL at embryonic day E10.5 to is very similar to the genes analysed by qPCR (Figure 3B 11.5. We applied PLA on FL sections at E12.5 to under- and Online Supplementary Figure S3). There is an increase of stand the temporal appearance of the two GATA1 com- expression of the transcription factors Ldb1, Gata1 and plexes in definitive blood cells. Figure 2 shows the com- Fog1 from P1 to P2 (together with E2A) and continuing to parison of the GATA1/LDB1 and GATA1/FOG1 complex- P3 for Gata1 and Fog1 (together with Klf1). Gata2, c-myb es, together with negative controls of GATA1, FOG1 or and c-kit expression is inversely correlated and follows the LDB1 single-primary antibody. Although FL tissue is com- same trend as Eto2 and Irf2bp2, with a decrease peaking at pact and single cells can be difficult to distinguish, clearly P3 followed by erythroid specific markers such as β-globin, some cells contained very dense GATA1/LDB1 PLA sig- Alas2 and Gypa and the transcription factor Lmo2. The nals when compared to surrounding cells (Figure 2A). result shows that the sorting method clearly separates the Close up images show that the signal co-localizes with the different stages of the erythroid cells. The significantly dif- DAPI staining (of note, those cells have little cytoplasm ferentially expressed genes (±0.6 log log two-fold change relative to the size of the nucleus) and that cells with no and P-value ≤0.05) between the populations is shown in signal (next to strong positive ones) include FL endothelial the Online Supplementary Table S3. Of note Ldb1 expres- cells which are expected to be negative for GATA1. A sim- sion presents a two-fold increase between P1 and P2, but ilar result was found in fetal aorta (not shown). Specific PLA is not included in the Online Supplementary Table S3 due to

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Figure 2. Proximity ligation assay of GATA1 complexes in E12.5 fetal liver tissue. (A) Proximity ligation assay (PLA) for GATA1/FOG1 and GATA1/LDB1 interactions was performed on sections of mouse fetal liver (FL) tissue at embryonic day 12.5 (E12.5), together with GATA1, FOG1 or LDB1 single-primary antibody negative controls. PLA protein interactions were visualized in red, and DAPI staining in blue was used to visualize the nucleus (scale bars, 50 μm in 20x and 40x). For each interaction zoom-in pictures corresponding to the white square area are also shown (scale bars, 20 μm in 63x). Z-stack images of each protein combination and fluorescent channels were projected by Maxi-Projection algorithm. (B) PLA for GATA1/LMO2 was performed as in Panel A using LMO2 antibodies replacing FOG1 antibodies. Scale bars represent 50 μm apart fromscale bars in zoom in picures bars which represent 10 μm.

1806 haematologica | 2020; 105(7) Temporal and spatial emergence of GATA1 complex the threshold parameters used in our study to detect the genes have erythroid differentiation bio-functions. Down- strongest differentially expressed genes. In P1 and P2, 65% regulated genes, (24% and 33%) show functions like lym- of the downregulated genes are enriched for e.g. neu- phocyte differentiation, B-cell differentiation and lympho- trophil degranulation, cytokine production and hemosta- cyte migration. At these stages (P3 to P4), cell cycle func- sis, i.e. genes crucial for other cell types (Figure 3D). The tions are suppressed as erythroid cells enter the terminal up-regulated genes (35%) represent essential functions for differentiation and proceed to enucleation. Next erythroid genes, e.g. erythrocyte homeostasis, porphyrin- GATA1/LDB1 PLA was applied on the four sorted popula- synthesis and cell cycle genes as cells at this stage are still tions (Figure 4A), showing high signals in P2 and P3. replicating. During mid- (P2 to P3) or late (P3 to P4) differ- Quantification confirmed that P2 had the highest density entiation, the majority (76% and 67%) of upregulated of GATA1/LDB1 interaction signals per nuclear area

A B C

Figure 3. Fetal liver cell sorting and RNA-sequencing data analysis. (A) Schematic description of erythroblast development. Fetal liver (FL) cells are sorted into four populations based on membrane markers TER119 and CD71. Different stages of erythroblasts are indicated into P2 to P4 populations. The C-KIT positive cell population (P1, CD71–/TER119–) represents precursors or other lineage cell types present before the initiation of erythroblast maturation. Proerythroblasts/colony forming unit-erythroid [CFU-E] cells) express high level of CD71 and low level of TER119 (P2, CD71+/TER119–). Following erythroblast maturation, TER119 expression increases (P3, CD71+/TER119+). When the differentiation reaches the orthochromatic stage, they loose CD71 expression (P4, CD71–/TER119+). Pro: immature proerythroblast; Baso: basophilic erythroblast; Poly: polychromatic erythroblast; Orth: orthochromatic erythroblast; Ret: reticulocytes; Ery: erythrocyte. Gray bars represent the changes of c-Kit, Cd71 and Ter119 gene expression. The darker color of the bar represents higher expression for the indicated gene. (B) Quantitative PCR on indicated genes in E12.5 or E13.5 sorted FL cells (E12.5 and E13.5 were used as duplicates) as described in Panel A. Relative expression values are calculated by comparing to control gene Rnh1. (C) Principle component analysis of RNA-sequencing data from two replicates of each P1 to P4 sorted FL cells as described in (A) (D) The total number of down-regulated or up-regulated differentially expressed genes (DEG) are shown for each comparison in a Volcanoplot, in which x-axis represents log two-fold change and y-axis represents log 10 adjusted P-value. Significant DEG are shown in blue for down-regulated and red for up-regulated. Gray dots represent the non-significant genes. The ratio of down- or up-regulated DEG in each comparison is shown in pie-chart inserted in the Volcanoplot. Top 20 gene ontol- ogy terms for down- or up-regulated DEG in each comparison are shown.

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(Figure 4B). Interaction between the LDB1 and GATA1 undifferentiated states (Figure 5C). In MEL cells, the LDB1 proteins appears already shortly before the proerythrob- complex binds its target genes during erythroid differenti- 23 last stage. It is most abundant in the P2 population (most ation, e.g. the α- and β-globin locus bind the LDB1 com- likely colony forming unit-erythroid [CFU-E] cells) and plex in differentiated cells resulting in upregulation,24-27 due basophilic erythroblasts but decreases during the final to the loss of the repressor ETO2 (encoding by Cbfa2t3) stages of differentiation in vivo. We applied PLA to detect from the complex.9 We quantified the PLA signals for the interaction between GATA1/FOG1, GATA1/LDB1, GATA1/LDB1, LDB1/LMO2 and LDB1/ETO2 interactions GATA1/TAL1, LDB1/LMO2 and LDB1/ETO2 in MEL (Figure 5D-F) and confirmed not only an increase of both cells, which mimic FL cells. Uninduced MEL cells repre- GATA1/LDB1, and LDB1/LMO2 interactions upon differ- sent proerythroblasts, i.e. part of the P1 and P2 population, entiation but further confirmed that the LDB1/ETO2 while induced MEL cells represent P3 and further differen- interaction is lost during differentiation in MEL9 (Figure tiated populations.9, 28,29 Figure 5A shows the detection of 5F), supporting its role as a negative regulator during ery- the GATA1/FOG1 interaction by PLA using single-prima- throid differentiation where ETO2 and IRF2BP2 with the ry antibody and secondary antibody alone as controls. NCOR1/SMRT co-repressor complex suppress the expres- Quantification of the PLA signals in nuclei show a signifi- sion of typical erythroid genes such as Klf1 which is need- 8,26 cant increase of GATA1/FOG1 interaction after MEL cell ed to express β-globin and Gypa genes. differentiation. An additional negative control experiment In conclusion, the GATA1/LDB1 complex starts to be to further demonstrate the specificity of the PLA assay formed just before the proerythroblast stage and activates was a TAL1/FOG1 interaction which is known not to be erythroid specific genes of erythroid differentiation in vivo, formed.5 Quantification of the different PLA signals con- when it looses ETO2. firmed the absence of TAL1/FOG1 interaction detection in MEL cells and is comparable to the one of the single probe LDB1 KD results in loss of the erythroid cell GATA1 only control, thereby supporting the specificity of population positive PLA signals (Figure 5B). This control on non-inter- We examined the importance of the GATA1/LDB1 com- acting highly expressed proteins (TAL1 and FOG1 in MEL plex in fetal erythropoiesis at E12.5 by three independent cells)5 also ruled out a potential threshold effect of PLA i.e. (partial) KD rather than a (lethal) KO using anti-LDB1 where ligations may be more likely when the relevant TF shRNA (shLDB1_1 and shLDB1_2) and an anti-GATA1 partners are expressed highly. Similar quantification of shRNA (shGATA1). Treatment with an empty vector PLA signals was performed in the cytoplasm (Figure 5B). It pLL3.7 shRNA or scrambled shRNA (Scr) was used as the was much lower than that observed in the nucleus and controls. The level of LDB1 or GATA1 protein relative to does not increase upon differentiation as seen in the valosin containing protein (VCP) decreased in the FL cells nuclei. Of note the increase of signal was not due to an from D1 to D3 in the KD (Online Supplementary Figure S4). increase of signal volume between the differentiated and On D3, the cells were sorted by fluorescence-activated cell

A B

Figure 4. GATA1/LDB1 proximity ligation assay on sorted fetal liver cells. (A) GATA1/LDB1 proximity ligation assay (PLA) on the four sorted cell populations as described in Figure 3A. PLA signal is in red and nucleus in blue. Scale bar represents 20 μm. (B) Quantification of GATA1/LDB1 PLA signals from three biological replicates (total number of dots in one cell vs. nucleus area) and boxplot comparison among the sorted four cell populations. ****Indicates the significance (P≤0.0001) between any of two cell populations. The statistical significance is determined by Kruskal-Wallis test (Tukey method).

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B C

Figure 5. Proximity ligation assay on GATA1 complexes in MEL cells. (A) GATA1/FOG1 proximity ligation assay (PLA) on non-induced and induced MEL cells. PLA signal representing protein-protein interaction in white in a black background and in red in E merged pictures together with DAPI in blue. GATA1 or LDB1 antibody alone was also performed as negative controls. All scale bars represent 20 μm. The significance from three biological replicates was analyzed with Kruskal-Wallis test as ****: P≤ 0.0001 and *: P≤0.05. The statistical significance is determined by Kruskal- Wallis test (Tukey method). (B) PLA detection of GATA1/FOG1 and GATA1/TAL1 interaction compared to non-existing interaction FOG1/TAL1 in MEL non-induced cells. The PLA signal is separated into relative ratio in nucleus or cytoplasm. ****Indicates the significance (P≤0.0001) between F indicated interactions and is determined by Kruskal-Wallis test (Tukey method). (C) The volume of individual GATA1/LDB1 PLA signals from 53 MEL non-induced and 56 induced cells is calculated following deconvolution and shown in boxplot. P-values of both statistical analysis of parametric (t-test) and non-parametric (Mann- Whitney) are 0.827 and 0.674, respectively. (D) GATA1/LDB1 PLA on MEL cells as in (A) (E) LDB1/LMO2 PLA on MEL cells as in Panel A. Panel F: LDB1/ETO2 PLA on MEL cells as in Panel A.

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sorting (FACS) using TER119 and CD71 expression and GATA2 is present during early erythropoiesis and binds the relative ratio normalized versus pLL3.7 control (ctrl), in to the Gata1 gene to activate its expression.19 Gata1 each population is shown in the Online Supplementary expression in turn represses Gata2 expression via the Figure S4B. LDB1 protein decreased by ~60% at D3 FOG1/MeCP1 complex, while activating its own expres- (54.3%, for shRNA#1 and 64.3% for shRNA#2 compared sion. This "GATA switch" represents a forward drive to ctrl) which resulted in a ±50% decrease in the P2, P3 towards late stage erythroid differentiation through and P4 population of cells (see above). In three additional changes in gene expression.19,31 GATA2 regulates impor- LDB1 KD, a pLL3.7 plasmid containing EGFP was used to tant proliferation genes of stem or progenitor cells where- determine the RNA levels in GFP+ cells versus the total cells as GATA1 also regulates the final erythroid fate through (“All cells”). As expected, the “All cells” result shows a the expression of erythroid specific genes. A similar similar ratio in each population as was observed in Figure process operates early during embryonic development. 3A. The GFP+ cells representing the really infected cells, GATA2 is present before GATA1 during ES cells differen- showed a strong KD effect in P2 to P3 compared to pLL3.7 tiation to the hematopoietic lineage. The GATA2/LDB1 control, when the level of LDB1 appears highest (Figure complex is present in very low concentration binding a 3A). The GATA1 KD showed a decrease of GATA1 pro- small set of hematopoietic specific transcription factors in tein of ~43.5% at D3 (relative to Scr ctrl) resulting in a a co-factor dependent manner (PK, CA-S and FG, manu- ±40% decrease in the P3 population while majority of script in revision). Unfortunately, the low level of GATA2 cells are P1. This result is consistent with the phenotype and poor quality of its antibodies prevent a PLA signal observed in inducible Gata1 KO mice showing an over- although the GATA switch must start early as we identi- representation of cells in P1 and an under-representation fied the GATA1/LDB1 and LDB1/E2A complexes already of cells in P3 and P4).28 The difference of the KD effect in in D4 EB. PLA does not show which cell population con- LDB1 and GATA1 therefore correlates with their expres- tains the GATA1/LDB1 and LDB1/E2A complexes in D4 sion in those populations (Figure 3B). It is impossible to EB, but it is known that LDB1 is expressed in BL-CFC.10 conclude whether the decrease in the LDB1 complex has The cells expressing GATA1 and LDB1 in D4 EB are differ- an effect before the proerythroblast stage, because the entiating hematopoietic cells. We know from ChIP-seq number of progenitors committed to the erythroid lineage data that the two factors already form a complex at this in the P1 is not known, but the 40-50% decrease in P2 to stage targeting a core set of genes enriched for categories P4 suggests that there is a decrease in the number of related to differentiation/quantity of blood/hematopoietic proerythroblasts that is propagated to the later cell com- progenitor cells (Kolovos et al., manuscript in preparation). partments. Of note, the cells (P2 to P4) were strongly Moreover, we described in Mylona et al. 201310 that blast affected by the KD of LDB1 or GATA1 and were dying. colony-forming cells deficient for Ldb1 (i.e. Flk1+ cells sorted four days after ES cell differentiation) are unable to dif- Discussion ferentiate to the hematopoietic lineages with a severe reduction in the number of blast colony forming cells and Our study shows a modulation of the levels of the pro- their failure to give rise to blast colonies. tein complexes from the early stages of erythroid differen- These cells appear between ES cell differentiation D3.75 tiation of the systems that we used (erythroid differentia- to D4.25 expressing vascular endothelial growth factor tion from embryonic stem cells, MEL cells, primary ery- receptor-2 (VEGFR2, i.e. FLK1). The absence of LDB1 throid cell populations from mouse FL and FL tissue sec- results in less BL-CFC and failure to generate hematopoi- tions). These diverse systems represent different stages etic and endothelial lineages.10 Of its targets Gata2, and therefore heterogeneous systems of hematopoietic Scl/Tal1, Runx1 and Gif1b are down-regulated showing development and differentiation. They show that the that the complex is essential for activation of early embry- same complex is formed at these quite different stages, onic hematopoiesis in agreement with our observation although these experiments do not directly show that the here that the GATA1/LDB1 interaction already takes place formation of the complex is essential at all these stages at this early stage. It has been shown that a KO of Gata1 and one system might not directly extrapolate to the in ES cells did not affect the formation of clonogenic pro- other. However previous data using KO or KD experi- genitors in chimeric in vitro differentiation, and Gata1 KO ments of individual components of the complex lead to colonies contained phenotypically normal macrophages, defective hematopoiesis/erythropoiesis.9,10,29 We therefore neutrophils and megakaryocytes,3,4 suggesting that GATA1 conclude that GATA1/LDB1 complex formation is essen- is not yet essential at this early stage. Importantly GATA2 tial in these diverse systems and provide novel insight in is essential for the generation of FLK1+ BL-CFC during GATA1 complexes. The result describes the sequential in vitro ES cell differentiation32 and Gata2-/- embryos die at emergence of GATA1/LDB1, GATA1/FOG1 and E11.5 with severe anemia.30 LDB1/E2A complexes in early stage ES cell differentiation, LDB1 IP experiments in undifferentiated ES cells show suggesting dynamic changes of the complexes and their that a GATA1/LDB1 complex is absent, but appears at low function taking place during the blast colony-forming cell levels in nuclear extracts (NE) from D4 and D5 ES cell EB (BL-CFC) stage similar to the hemangioblast stage in vivo. (Online Supplementary Figure S1C-D). The expression of GATA1 as part of the LDB1 complex is absent in undiffer- FOG1, LDB1 and GATA1 increases from D4 to D5, but is entiated ES cells and only appears after a few days of dif- still very low and the binding partner LDB1 could not ferentiation, while GATA2 is expressed and is part of the detected by IP in a GATA1 pulldown (Online LDB1 complex in the very early stages of differentiation Supplementary Figure S1). The E2A protein was detected in where it activates GATA119 in a feed forward type system the LDB1 IP only at D5, but again the band is very weak. (Kolovos et al., in revision), which is in agreement with the We also applied size-exclusion chromatography to distin- data on the function of GATA1 and GATA2 in mice or dif- guish the different GATA1 complexes. The expression of ferentiating ES cells.4,30 LMO2 protein increases at D4 (Online Supplementary Figure

1810 haematologica | 2020; 105(7) Temporal and spatial emergence of GATA1 complex

S1B), most of it is in the fractions of 37-39, but some is remains in the cytoplasm is a possibility. The extensive set present in fractions 22-24 indicating that some complex of complementary experiments that would aim at study- has formed. In MEL cells, i.e. a much later stage of devel- ing the existence of interactions in the cytoplasm, such as opment and differentiation, most LMO2 is in the fractions cellular compartment fractionation followed by IP would overlapping with LDB1 (Online Supplementary Figure S1B), be similarly questionable, as (like for PLA) it would be dif- showing they are in the same complex consistent with ficult to exclude the possibility of the nuclear extract frac- previous results.8 Although the expression of GATA1 and tions leaking out in the cytoplasmic fractions. We per- partners is low at D4-5 ES cell differentiation, we could formed an additional control by quantifying the PLA sig- detect the formation of the different complexes by PLA nals in the cytoplasmic area of MEL cells for the positive due to its ability to detect 102-fold or 106-fold lower pro- GATA1/FOG1 and the non-existing TAL1/FOG1 interac- tein concentrations than ELISA or WB, respectively.33 tions and associated negative controls (single probes and GATA1/FOG1 regulates genes that are less important secondary antibodies only), and observed that non-exist- for erythroid specific functions in the undifferentiated ing TAL1/FOG1 interaction detection presents a lower stage, e.g. pro-erythroblasts. We originally postulated a level to the existing GATA1/FOG1 and GATA1/TAL1 in switch from GATA1/FOG1, repressing alternative lineage the cytoplasm (Figure 5B). These differences suggest the genes to GATA1/GFI1B repressing proliferation related existence of a certain level of transcription factor interac- genes during differentiation.5 In MEL non-induced cells, tion in the cytoplasmic fraction and the amount of signal GFI1B and LDB1 can also be detected in an ETO2 pull- in TAL1/FOG1 sets the level of background of the PLA down, indicating that LDB1 complexes containing ETO2 technique. It should also be noted that the level of PLA sig- and/or GFI1B proteins suppress archetypical erythroid nals from different interactions cannot be compared genes primed for the onset of terminal erythroid differen- directly, since PLA is dependent on antibody quality. tiation.26 Indeed, RNA-seq data shows that Cbfa2t3 Therefore, like other immuno-based technic PLA present (encoding ETO2) and Irf2bp2 expression decreased from a certain level of background, easily quantifiable by using P1 to P2 suggesting these genes mainly function in P1 to single probe, secondary antibodies only, protein mutant P2, similar to the stage of non-induced MEL cells. and/or non-existing interaction detection. PLA can detect Meanwhile, Klf1 increased its expression and reached the an interaction up to a 40 nm distance36 and enables super peak at P3, together with the increase of typical erythroid high resolution immunofluorescence microscopy, which specific genes (Online Supplementary Figure S3). This result can distinguish molecules at a similar distance37. Although indicates that clear erythroid differentiation starts at P2. PLA cannot detect real-time protein-protein interaction, Our PLA result on sorted FL cells show that the quantification of PLA signals of different GATA1 complex- GATA1/LDB1 interaction peaks in CD71+/TER119– cells at es in sequential stages of ES cell differentiation improves a relatively early stage of erythropoietic differentiation, our understanding on the temporal changes of these com- like undifferentiated MEL cells, when many erythroid plexes. genes are still suppressed, until ETO2 disappears from the This study therefore reveals that PLA is a powerful tool complex turning on typical erythroid genes. Other LDB1 to examine dynamic protein/protein interactions and their complex regulated genes such as c-myb remain suppressed, dynamics in differentiating erythroid cells and demon- because they no longer bind the (activating) LDB1 com- strates that it provides an excellent alternative for cells in plex through an as yet unresolved mechanism which the abundance of proteins is too low to perform (Giraud et al., unpublished data). standard co-IP experiments. Our study revealed that PLA We show that PLA can detect endogenous level of can be used to detect very low amount of essential GATA1 GATA1/LDB1 and GATA1/FOG1 interactions in FL tissues complexes emerging at early time point of ES cell differen- and we located the GATA1/LDB1 interaction in a specific tiation and later on FL tissue, the site of definitive erythro- cell type in situ. Whether it corresponds to E7.5 yolk-sac poiesis. In addition we show that the increase followed by derived primitive cells, E8.5/E9 yolk sac and placenta a decrease of expression of GATA1 and LDB1 affects a derived intermediate erythroid-myeloid,34,35 or HSC number of genes differentially, for example the expression derived definitive progenitors is still an open question. of the c-myb gene which is regulated by the LDB1 com- From PLA images a number of transcription factor com- plex15 decreases during differentiation, while the expres- plexes seem to be located outside the nuclei. The studied sion of the β-globin genes, which are also dependent on factors are certainly formed in the cytoplasm and travel to the LDB1 complex increases. Further studies will be need- the nuclei; whether a fraction of interacting factors ed to understand how these differences are regulated.

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